Of course, biosafety was a primordial part of our project, given that we have been working on genetically modified organisms. We conducted a reflexion about the different ways to make our project the safest for us and for the environment in the case it would become a concrete application. It was our will to push the thinking to fully developed biosafety measures for our project. In this way, we wished to demonstrate our anticipation of the concretisation of our product.

The safety applications that we put to light can be grouped in two categories: the biological safeties, and the mechanical safeties. The biological area focuses on all the means that can be integrated in our biosensor, being genetic modifications or epigenetic mechanisms that will influence the cell behaviour if conditions are required. Those are taking a significant amount of time to create in the lab, and though we did research and develop the idea, we did not have enough time in the iGEM competition to implement them. We will however present them in this part to show our reflexion.

We thought of several tracks to follow for our biological safeties:

• • Kill switch: The kill switch is a well-known method to prevent a micro-organism culture from contaminating its environment. It can have various forms ( toxin/antitoxin system which will be further developed, genetic switch-off, genetically engineered logical gate) and it allows to induce cell death as soon as the condition that activates the kill switch is fulfilled. It is one of the best option we can use in our system to allow a strict biological control of our micro-organism.

• • Bacteria traceability: through a tag (fluorescence, radioactivity, or just genetic tagging of a constitutive compound) we could keep track of the micro-organism when it would come to a leak in the open environment. This method would allow to follow, contain and eventually prevent any further contamination of the micro-organism – bacteria for Quantifly – in a dangerous or harmful way for the environment.

• • Cellular cycle inactivation: with the inactivation of the micro-organism’s cellular cycle, we can prevent it from replicating and thus we are sterilizing it. This method has the advantage to prevent colonies proliferation. We can also keep control of the population in our product, but it involves a heavy manipulation in the lab to inactivate a whole cell culture and the lifespan of such cells is really short as it is a matter of minutes before cell death would activate. Though this method could be a mean of control in case of environmental contamination, it is not the appropriate technique to use in our project.

• • ATP production limitation: This track is thought as a genetic engineering to have our micro-organism produce less ATP, producing just enough energy to make sure it can survive but not develop (as the cell replication requires important amounts of energy). By limiting the ATP production in the cell, we are ensuring it won’t be able to go through its cycle, and we can control again the population growth. However it will certainly put micro-organisms in stress conditions, which cannot guarantee the functioning of our biosensor.

• • Toxin/anti-toxin system: the aim of this method would be to have our micro-organism produce a toxin to which it is not naturally immune, and in completion the medium in which the cultures are done provides the anti-toxin, allowing the micro-organism to survive in our controlled environment. As soon as the micro-organism escapes this environment, it will die from the intoxication. This method is one of the best we can have at the biological level since it makes our micro-organism dependant to the medium we are controlling and it is directly preventing any contamination in or from our cultures.

As biological safeties focus on the control and /or prevention of our micro-organism contamination, which is quite heavy to achieve in the given time for our project, the mechanical safety measures are, for some of them, simpler to input in the development of our product. They are directly linked to the mechanical aspect of the project, being the drone and its software. We thought of several options that could guarantee the control of the device and assure total safety for the manipulations it is designed to do. Thus, we will detail these different options and we will explain the reflexion behind the idea and how we could implement them on our device.

• • Organic detergent in tubes: the idea is to create an envelope around the micro-organism’s tube on the drone that will contain organic detergent. Thus, if the drone were to crash and the tubes break, the cultures would make contact with the detergent, leading to their complete destruction on-spot.

• • Injection system: another way of using the organic detergent is to create an injection system that would function on an altimeter and when an intense fall in altitude is detected, detergent would be injected in cultures, inducing the destruction of the culture.

• • Tubes’ material: as well as a crash procedure, we want to anticipate on this event by working on the cultures’ tubes material. The use of glass would be prohibited since the durability of such resource is not good at all in the case of a shock. The ideal material would be a strong and resistant plastic such as polyvinyl chloride (PVC) that can endure a several meters fall and the impact with the ground, while preventing cultures to spread in the environment.

• • Parachute: A simple safety we can input on the drone is a parachute that would be activated when altitude is falling too fast, preventing the drone to brutally crash on the ground.

• • Drone systems reboot for control loss: another issue we can come across is the failure of the systems of the drone, which can lead to a fall down. If such thing should happen, the presence of a reboot input (red button) can restart the drone while still in the air, allowing to retake control of the device quickly preventing an accident.

• • Global positioning system control: besides of falling, another issue for the drone can be to get out of the area defined for the measures, which could create a dysfunction in the drone piloting and/or flying abilities. A solution we thought is to input in the software a GPS detection of the drone to the area designed, and when the drone gets out of the zone, it will automatically come back into the measurement area.

The biosafeties as well as the mechanical solutions are compiling into a complex and safe device that can still be easily manipulated. The most careful way to do is to create safety measures in a layer format. The ideal is to concretise all of those safety leads, but we can basically work on a three-layer concept coordinating biological safeties such as a kill switch or a toxin/ani-toxin system coupled to an organic detergent injection system in case of falling as well as a parachute. The stacking of different safety measures is the best option given that it covers most of the risks we can face.